CN104302760A

CN104302760A - Method and device for isolation of non-fat cells from an adipose tissue

Info

Publication number: CN104302760A
Application number: CN201280068472.XA
Authority: CN
Inventors: L·R·黄; 施冰如; 廖智菁
Original assignee: Industrial Technology Research Institute ITRI
Current assignee: Industrial Technology Research Institute ITRI
Priority date: 2011-12-07
Filing date: 2012-12-06
Publication date: 2015-01-21
Also published as: HK1206060A1; TW201329231A; WO2013086199A1; JP2015500031A; KR20140147805A

Abstract

In accordance with an aspect of the present disclosure there is provided an apparatus for isolation of non-fat cells from an adipose tissue sample. The apparatus comprises a first sheet of material, a second sheet of material bonded to the first sheet of material, and a plurality of chambers defined between the first sheet of material and the second sheet of material, the plurality of chambers including a sample dissociation chamber including an inlet and an outlet, a waste collection chamber including an inlet in fluid communication with the outlet of the sample dissociation chamber, and a cell refinement chamber including an inlet in fluid communication with the sample dissociation chamber and an outlet.

Description

For being separated the method and apparatus of non-fat cell from fatty tissue

Background of invention

Biology with the many technology in medical science as cellular segregation, flow cytometry, raji cell assay Raji and cell therapy depend on disintegrated tissue to be separated respective cells.Treatment group tissue samples often relates to various motion, as chopping, washing, enzymic digestion, dissociate, hatch, mix, grumeleuse and fragment remove, and concentrated.In research laboratory, these actions are often manually carried out, and are being shifted from testing tube to testing tube in an open environment by sample.Described manual processes needs well-trained personnel and can cause potential pollution and infection risk to the manipulation of biological sample.

Recently, research and medical community are paid close attention to from fatty tissue isolated cell always.Have developed few techniques and remove fat tissue portions from patient or animal safety.Such as, tumescent liposuction technique and water jet liposuction technique have been widely used for removing fatty tissue from patient.Fatty tissue contains the adipocyte of depot fat and maintains other non-fat cell of tissue.The composition cell of fatty tissue, its effect and its interaction are not yet completely understood, and are active science and the theme of clinical study.

In order to study fatty tissue, may disintegrated tissue be needed and be separated composition cell.Described process may relate to release composition cell, remove fragment and undesired cell, concentrated also enrichment relevant cell, and washed cell.Described process can be effort, and may require well-trained operator, the setting of expensive equipment and have the laboratory of suitable biological safety measure.Described multiple control action also can cause the remarkable loss of relevant cell, thus makes the rare and separation difficulty of the cell that ubiquity is low and unreliable.In addition, when end user's sample, the risk of crossed contamination and infection may be significant.

Therefore need to have the method for being effectively separated relevant cell, cell especially except adipocyte from fatty tissue, and have and make to be separated non-fat cell from fatty tissue and become easy and the device of safety.

The bag comprising flexible plastic sheet is widely used in collecting, processing and store biological tissue samples, as peripheral blood, Cord blood, blood constitutent, blood plasma, marrow, liposuction aspirates etc.Bag has flexibility and inflatable advantage, and can change its internal volume to adapt to the sample of different volumes.In order to be conducive to sample preparation, exterior tubing is often used to connect, many indivedual bags of fluids to form a system.Described bag and pipeline system are widely used in sample preparation, such as blood part, cellular segregation etc.But owing to being integrated with more process action, therefore bag and pipeline system become rapidly heavy, be difficult to use and be difficult to manufacture.System becomes insulation covering tubulose, and becomes easy winding, and many parts dangle each other.In order to use described device, operator need high-caliber training and long-term experience to arrange this complicated apparatus.Operator also need to be also noted that and in the correct order various part are arranged on correct position.In addition, the manufacture of these devices and system can be difficulty and costliness, because multiple bag, parts and pipeline stretch section often must individually manufacture and then assemble.The assembling of described device may be labor-intensive, and can present the risk of leakage and pollution, the i.e. system fault.For clinical application, device is often that single uses, and its reliability is important.Intensive labour and plant failure risk are the major obstacles of these Conventional insulation cannula-like systems.

Summary of the invention

According to one side of the present disclosure, provide a kind of equipment for being separated non-fat cell from adipose tissue sample.Described equipment comprises the first material piece, is bonded to the second material piece of the first material piece and is defined in the multiple rooms between the first material piece and the second material piece, and described multiple room comprises: sample dissociation chamber, and it includes an inlet and an outlet; Waste collection room, it comprises the entrance be communicated with the outlet fluid of sample dissociation chamber; And cell refines room, it comprises the entrance be communicated with sample dissociation chamber fluid, and outlet.

According to some embodiments, sample dissociation chamber comprises screen filter further, and described screen filter comprises the hole of aperture between 70 μm and 300 μm.

According to some embodiments, described equipment comprises screen filter further, and described screen filter is included in cell and refines in room, comprises the hole of aperture between 20 μm and 50 μm.

According to some embodiments, sample dissociation chamber comprises the first screen filter further, described first screen filter comprises the hole with the first aperture, and wherein the cell room of refining comprises the second screen filter further, described second screen filter comprises the hole with the second aperture, and wherein the second aperture is less than the first aperture.

According to some embodiments, described equipment comprises the component that Quality control dissociation chamber, waste collection room and cell refine the fluid connection between room further.

According to some embodiments, the component controlling fluid connection comprises stopcock.

According to some embodiments, described equipment comprises flow rate control device further, and described flow rate control device is configured at least one of conciliating in exsolution liquid by cleaning solution and is incorporated in sample dissociation chamber, and has the outlet be communicated with sample dissociation chamber fluid.

According to some embodiments, described equipment comprises further for sample dissociation chamber and cell, of refining in room executes stressed component.

According to some embodiments, described equipment comprises downsteam processing facilities further, the outlet fluid that described downsteam processing facilities and cell refine room is communicated with and comprises at least one microfluidic device, described microfluidic device is configured to the fluid exported from the cell room of refining to be separated into the first solution and the second solution, described first solution has one or more relevant cells of the first concentration, described second solution has one or more relevant cells described of the concentration being less than the first solution, and wherein relevant cell comprises the non-fat cell be separated from adipose tissue sample.

According to one side of the present disclosure, provide a kind of aseptic and fatty tissue treatment system of substantial barrier.Described system comprises organized processing room, and it comprises entrance, outlet, and at least one screen filter, and described screen filter is arranged between the entrance of organized processing room and the outlet of organized processing room; Waste collection room, itself and organized processing room are included in same enclosed space, and described waste collection room comprises the entrance be communicated with the outlet fluid of organized processing room; And fragment removes one in room and sample collection room, described fragment is removed room and is comprised debris removal mechanism, and described sample collection room and organized processing room are included in same enclosed space, and are communicated with organized processing room fluid.

According to one side of the present disclosure, provide a kind of method processing adipose tissue sample in tissue processing system.Described method comprises and is incorporated in the first Room by pending adipose tissue sample by the ingress port of the first Room, process the adipose tissue sample in the first Room, and be transferred to the second Room by the outlet of cell by the first Room, the entrance by the second Room from the first Room, described second Room and the first Room are included in same enclosed space.

According to some embodiments, process adipose tissue sample comprises the adipose tissue sample that dissociates.

According to some embodiments, process adipose tissue sample comprises removes excessive fluid from the adipose tissue sample the first Room.

According to some embodiments, process adipose tissue sample comprises and uses cleaning solution to wash adipose tissue sample in the first Room.

According to some embodiments, process adipose tissue sample comprises and uses cleaning solution to wash adipose tissue sample in the first Room, and uses the adipose tissue sample that the solution that dissociates comprising at least one enzyme dissociates in the first Room.

According to some embodiments, the solution that dissociates comprises collagenase.

According to some embodiments, the solution that dissociates comprises collagenase, deoxyribonuclease and Unidasa.

According to some embodiments, the solution adipose tissue sample that dissociates that dissociates is used to carry out at about 37 DEG C.

According to some embodiments, described method comprises the screen filter removal fragment using and comprise in the second chamber further.

According to some embodiments, the aperture of screen filter is between 15 microns and 100 microns.

According to some embodiments, described method comprises further and being retained in the first chamber by sample, and by waste streams by comprise screen filter in the first chamber and the first Room the first outlet, transfer in the 3rd Room by the entrance of the 3rd Room, described 3rd Room and the first Room are included in same enclosed space.

According to some embodiments, described method comprises use microfluidic device enrichment non-fat cell mass further.

According to some embodiments, non-fat cell comprises stem cell.

According to some embodiments, described method comprises further from tissue processing system harvested cell.

According to some embodiments, described method is included in downsteam processing facilities further and processes cell, described downsteam processing facilities is communicated with the outlet fluid of the second Room and comprises at least one microfluidic device, described microfluidic device is configured to cellular segregation be become the first solution and the second solution, described first solution has the non-fat cell of the first concentration, and described second solution has the non-fat cell of the concentration being less than the first solution.

According to some embodiments, the cell of results is vascular stroma part cell.

According to one side of the present disclosure, provide a kind of for the equipment from fatty tissue isolated cell.Described equipment comprises the first material piece, is bonded to the second material piece of the first material piece and is defined in the multiple rooms between the first material piece and the second material piece, and described multiple room comprises: sample dissociation chamber, and it includes an inlet and an outlet; Waste collection room, it comprises and the entrance organizing the outlet fluid of dissociation chamber to be communicated with; And cell refines room, it comprises the entrance be communicated with sample dissociation chamber fluid, and outlet.

According to some embodiments, sample dissociation chamber comprises filter screen further.

According to some embodiments, described equipment comprises filter screen further, and described filter screen is included in cell and refines in room.

According to some embodiments, sample dissociation chamber comprises the first filter screen further, and described first filter screen comprises the hole with the first aperture, and wherein the cell room of refining comprises the second filter screen further, and described second filter screen comprises the hole with the second aperture.

According to some embodiments, the second aperture is less than the first aperture.

According to some embodiments, described equipment comprises flow rate control device further, and described flow rate control device is configured to the one of conciliating in exsolution liquid by cleaning solution and is incorporated in sample dissociation chamber, and has and the outlet organizing dissociation chamber fluid to be communicated with.

According to some embodiments, described equipment comprises further for sample dissociation chamber and cell, of refining in room executes stressed component.Described component can be arranged between the first material piece and the second material piece.Described component can be arranged on around the first material piece and/or the second material piece.

According to some embodiments, described equipment comprises downsteam processing facilities further, the outlet fluid that described downsteam processing facilities and cell refine room is communicated with and comprises at least one microfluidic device, described microfluidic device is configured to the fluid exported from the cell room of refining to be separated into the first solution and the second solution, described first solution has one or more relevant cells of the first concentration, and described second solution has one or more relevant cells described of the concentration being less than the first solution.

According to one side of the present disclosure, provide a kind of fatty tissue treatment system of substantial barrier.Described system comprises fatty tissue treatment chamber, and it comprises entrance, the first outlet, the second outlet, and at least one filter screen, and described filter screen is arranged between the entrance of organized processing room and first of organized processing room export; Waste collection room, itself and organized processing room are included in same enclosed space, and described waste collection room comprises the entrance be communicated with the first outlet fluid of organized processing room; And fragment removes one in room and sample collection room, described fragment is removed room and is comprised debris removal mechanism, and described sample collection room and organized processing room are included in same enclosed space, and are communicated with the second outlet fluid of organized processing room.

According to some embodiments, described system comprises fluid volume measuring chamber further, and itself and fatty tissue treatment chamber are in same enclosed space, and the outlet comprising entrance and be communicated with the inlet fluid of fatty tissue treatment chamber.

According to some embodiments, the entrance of fluid volume measuring chamber and the outlet of fluid volume measuring chamber comprise vacuum breaker separately.

According to some embodiments, the first outlet comprises with the second outlet the stopcock be communicated with fatty tissue treatment chamber fluid and exports.

According to one side of the present disclosure, provide a kind of method from fatty tissue isolated cell.Described method comprises to be introduced in organized processing room by the ingress port of organized processing room by pending sample, the sample in treatment chamber is organized in process, and be discharged into sample storage chamber by the outlet of cell by organized processing room, the entrance by sample storage chamber from organized processing room, described sample storage chamber and organized processing room are included in same enclosed space.

According to some embodiments, described method comprises further and is retained in organized processing room by cell sample, and by waste streams by be included in screen filter in organized processing room and organized processing room the first outlet, transfer in waste collection room by the entrance of waste collection room, described waste collection room and organized processing room are included in same enclosed space.

According to some embodiments, described method comprises further extracts cell sample from tissue processing system.

According to some embodiments, described method is included in downsteam processing facilities the cell sample processing described extraction further, described downsteam processing facilities is communicated with the outlet fluid of sample storage chamber and comprises at least one microfluidic device, described microfluidic device is configured to the cell sample of extraction to be separated into the first solution and the second solution, described first solution has one or more relevant cells of the first concentration, and described second solution has one or more relevant cells described of the concentration being less than the first solution.

According to some embodiments, process is organized the tissue sample in treatment chamber to comprise will cleaning soln to be organized to be incorporated in organized processing room.

According to some embodiments, process organizes the tissue sample in treatment chamber to comprise further to be dissociated by tissue solution to be incorporated in organized processing room.

According to one side of the present disclosure, provide a kind of for the method from fatty tissue isolated cell.Described method comprises pending suction lipectomy matter sample to be incorporated into and comprises in the room of net, excess fluid is removed from sample, make the sample that dissociates by least one net to remove adipocyte and fragment, and substantially removed from sample by red blood corpuscle, and use microfluidic device by sample concentration at least three times.

According to some embodiments, described method comprises use washing soln washing sample further.

According to some embodiments, the solution that dissociates that described method comprises further with comprising at least one enzyme mixes and hatches sample.

According to some embodiments, described microfluidic device comprises multiple microfluidic channel, and described microfluidic channel has the degree of depth of substantially constant and at least one size is less than 750 microns, and wherein at least two microfluidic channel are arranged on a surface of substrate.

According to some embodiments, the solution that dissociates comprises collagenase, and wherein sample reconciliation exsolution liquid is hatched between about 20 DEG C and about 40 DEG C.

According to some embodiments, the solution that dissociates comprises collagenase and deoxyribonuclease, and wherein sample reconciliation exsolution liquid is hatched at about 37 DEG C.

According to some embodiments, microfluidic device comprises the post being configured to sample separation be become non-fat karyocyte part further.

According to some embodiments, microfluidic device is configured to use washing soln washing non-fat cell and substantially remove the enzyme dissociated in solution.

According to one side of the present disclosure, provide a kind of fatty tissue treatment system of substantial barrier.Described system comprises: sample washing and dissociation chamber, and it comprises three ingress ports, the first outlet port and the second outlet port, and is arranged on three ingress ports and the net between the first outlet port and the second outlet port; Grumeleuse reduces room, it comprise wash with sample and inlet attack that the first outlet port fluid of dissociation chamber is connected, outlet, and be arranged on inlet attack and net between exporting; For separating of cell and the reservoir removed of further fragment, it has the entrance that the outlet fluid that reduces room with grumeleuse is communicated with; Waste solution collecting chamber, it has the entrance washing with sample and be communicated with the second outlet port fluid of dissociation chamber; And microfluidic device.

According to some embodiments, described microfluidic device comprises multiple microfluidic channel, and described microfluidic channel has the degree of depth of substantially constant and at least one size is less than 750 microns.

According to some embodiments, at least two in described multiple microfluidic channel are disposed on a surface of substrate.

According to some embodiments, sample washing and dissociation chamber, each minimizing in room, reservoir and waste solution collecting chamber of grumeleuse include in same packing.

Accompanying drawing is sketched

Accompanying drawing is not intended to draw in proportion.In the drawings, each identical or intimate identical parts illustrated in different figures are by identical numeral.For clarity object, in each width figure, be not that all parts all mark.Unless otherwise instructed, otherwise all figure all should think schematically.In the drawings:

Figure 1A is the schema of the method according to an embodiment of the present disclosure;

Figure 1B is the schema of the method according to an embodiment of the present disclosure;

Fig. 2 A is the schematic diagram of the sample processing device according to an embodiment of the present disclosure;

Fig. 2 B is the schematic diagram of the flow rate control device according to an embodiment of the present disclosure;

Fig. 2 C is the schematic diagram of the flow rate control device according to an embodiment of the present disclosure;

Fig. 2 D is the schematic diagram of the sample processing device according to an embodiment of the present disclosure;

Fig. 2 E is the schematic diagram of the sample processing device according to an embodiment of the present disclosure;

Fig. 2 F is the schematic diagram of the sample processing device according to an embodiment of the present disclosure;

Fig. 2 G is the schematic diagram of the sample processing device according to an embodiment of the present disclosure;

Fig. 3 A is the front view of the sample processing device according to an embodiment of the present disclosure;

Fig. 3 B is the isometric view of the sample processing device of Fig. 3 A;

Fig. 3 C is the decomposition view of a part for the room of the device of Fig. 3 A;

Fig. 3 D is the decomposition view of a part for the room of the device of Fig. 3 A;

Fig. 3 E is the side cross-sectional view of a part for the room of the device of Fig. 3 A;

Fig. 3 F is the front view comprising the sample processing device of optional clip of Fig. 3 A;

Fig. 4 is the front view of the sample processing device according to an embodiment of the present disclosure;

Fig. 5 A is the front view of the room of sample processing device according to an embodiment of the present disclosure;

Fig. 5 B is the side cross-sectional view of a part for the room of Fig. 5 A;

Fig. 6 A is the front view of the room of sample processing device according to an embodiment of the present disclosure;

Fig. 6 B is the side cross-sectional view of the room of Fig. 6 A;

Fig. 7 is the side cross-sectional view of the room of sample processing device according to an embodiment of the present disclosure;

Fig. 8 A is the front view of the room of sample processing device according to an embodiment of the present disclosure;

Fig. 8 B is the decomposition view of the room of Fig. 8 A;

Fig. 9 A is the front view of the room of sample processing device according to an embodiment of the present disclosure;

Fig. 9 B is the decomposition view of the room of Fig. 9 A;

Figure 10 A is the front view of a part for sample processing device according to an embodiment of the present disclosure;

Figure 10 B is the decomposition view of a part for the sample processing device of Figure 10 A;

Figure 11 is the front view of the sample processing device according to an embodiment of the present disclosure;

Figure 12 is the front view of the sample processing device according to an embodiment of the present disclosure;

Figure 13 A is the front view of the sample processing device according to an embodiment of the present disclosure;

Figure 13 B is the cross sectional view of a part for the room of the device of Figure 13 A;

Figure 13 C is the cross sectional view of a part for the room of the device of Figure 13 A;

Figure 14 is the front view of the sample processing device according to an embodiment of the present disclosure;

Figure 15 A is the diagram of a part for the microfluidic device comprised in embodiments more of the present disclosure;

Figure 15 B is the diagram of the microfluidic device comprised in embodiments more of the present disclosure;

Figure 15 C is the diagram of the microfluidic device comprised in embodiments more of the present disclosure;

Figure 15 D is the diagram of the microfluidic device comprised in embodiments more of the present disclosure;

Figure 15 E is the diagram of the microfluidic device comprised in embodiments more of the present disclosure;

Figure 16 is the front view of the sample processing device according to an embodiment of the present disclosure;

Figure 17 is the diagram of the microfluidic device comprised in embodiments more of the present disclosure;

Figure 18 A is the photo of the fluid processed in embodiment of the present disclosure; And

Figure 18 B is the photo of the fluid processed in an embodiment of the present disclosure.

Describe in detail

Application of the present disclosure is not limited to the details of that set forth in following explanation or graphic middle institute diagrammatic structure and the layout of parts.The disclosure can realize other embodiment and differently can put into practice or carry out.And wording used herein and term are for purpose of explanation and should be considered as restrictive.Use " comprising ", " comprising " herein, " having ", " contain ", " relating to " and its version mean to contain the project and its counterpart and other project that listed thereafter goes out.

As used herein, term " sample ", " tissue sample ", " fat sample " and " fatty tissue " can comprise adipose tissue sample, such as liposuction aspirates, and described term is used interchangeably.。

As used herein, term " microfluidic device " can refer to have at least one fluid channel and to be formed on a surface substantially and at least one interconnection size is less than the device of about 1mm, and described surface can be substantially smooth or bending.Described interconnection size can be width or the degree of depth of such as passage.

Have been found that and need that there is a kind of method for being effectively separated relevant cell from fatty tissue, and have and make from fatty tissue isolated cell easily and the device of safety.For studies and clinical application, have been found that the various motion needing to process adipose tissue sample becomes streamlined, make personal errors to minimize like this.In addition, have been found that importantly, adipose tissue sample is processed in the environment of " isolation " substantially, wherein provide barrier to avoid such as being contacted or fluid contact with outside atmosphere and/or operator's direct physical by unfiltered airflow to isolate fat sample, thus minimize or avoid to pollute and infection risk.Also have been found that and need the system that has for fatty tissue process and device, wherein many parts and compartment are integrated into single type, to provide the environment of substantial barrier for sample.Preferably, be easy to use, be easy to manufacture and there is low failure risk for the system of fatty tissue process and device.For many clinical and research application, also may preferably device and system be aseptic with disposable with any part that adipose tissue sample directly contacts.

Many aspects of the present disclosure and embodiment provide a kind of method for being separated some composition cell mass from adipose tissue sample.Other side of the present disclosure and embodiment provide a kind of device realizing the described method for being separated some composition cell mass from adipose tissue sample by integrated, fairshaped, safe and wieldy mode.

Many aspects of the present disclosure and embodiment provide a kind of integrating device, it comprises multiple compartment for adipose tissue sample process, and described compartment can include but not limited to be constructed and arrange the compartment for following: sample collection, washing, layering, mixing, heating, cooling, filtration, digestion, storage, fluid transfer and manipulation, cell marking, sample preparation, dissociate, waste streams collection, grumeleuse removal, fragment removal, cell concentration, cell enrichment, cellular segregation, cell incubation, growth, cultivation, differentiation, amplification etc.Also can comprise for such as controlling fluid flow rate object between compartment and carry out establish valve according to the integrating device of embodiment of the present disclosure.Described device is applicable to integrated for multiple actions of adipose tissue sample process and make it become streamlined, described action case is as from fatty tissue isolated cell, and multiple function can be conducive to, as enzymic digestion, fatty tissue dissociate, wash, the removal of waste collection, fragment, cell concentration, use antibody labeling, use magnetic beads mark, cell amplification etc.Described device can be particularly useful for security, is easy to usability and is easy to the important application of manufacturing.Aspects more of the present disclosure and embodiment comprise the method using described device.

An embodiment for the method from fatty tissue isolated cell of the present disclosure include but not limited to dissociate fatty tissue, release composition cell, collect the cell discharged, and remove fragment of tissue.Described method can be included in the action that before tissue dissociates, fatty tissue is clean further.Fatty tissue cleaning action can comprise from adipose tissue sample removal or discharge undesired or excessive fluid.Described undesired fluid can comprise blood, body fluid and tumescent solution.Fatty tissue cleaning action can comprise the cleaning of use cleaning solution or washing fatty tissue further.Described method can comprise one or more actions of the cell that enrichment or purifying discharge further.In addition, described method can comprise further from described process collection waste streams.Another embodiment for the method from fatty tissue isolated cell of the present disclosure includes but not limited to remove excessive fluid from fatty tissue, and dissociate fatty tissue and release composition cell, and remove undesired cell and fragment.Described method can comprise further following in one or more: washing adipose tissue sample, concentrated relevant cell, washing relevant cell and use such as antibody to carry out immunity separation.In one embodiment, concentrated and/or washing relevant cell can use at least one microfluidic device to carry out.In another embodiment, one or more action can adopt centrifugal.In another embodiment, one or more action can utilize tubular fibre to carry out.

In an embodiment of the present disclosure, provide a kind of method for being separated non-fat cell mass from fatty tissue.Described method includes but not limited to remove excessive fluid from fatty tissue, fatty fatty tissue is washed with buffered soln, use such as ultrasonic wave or the solution disintegrated tissue of dissociating containing enzyme, remove adipocyte, free oil, matrix fiber and fragment of tissue, reduce red blood corpuscle, and enrichment relevant cell.Cell enrichment action can use whizzer, strainer or microfluidic device to realize.Described method can comprise further following in one or more: lymphopenia, cell washing and immunity be separated.Excessive fluid is removed from fatty tissue, fatty tissue is washed with buffered soln, dissociate fatty tissue, remove adipocyte, free oil, matrix fiber and fragment of tissue, and the action of cell washing can use such as gravity settling, strainer that is centrifugal and/or that comprise screen filter carries out.

Figure 1A illustrate generally be designated as 100, for being separated the schema of an embodiment of the method for non-fat cell mass from fatty tissue.In liposuction procedures process, swelling fluid is often introduced patient and is minimized to make blood loss, tissue is compacted, and provides toponarcosis.Tumescent solution can containing the lignocaine and 1: 1 of 0.05%, the suprarenin of 000,000 concentration.Described method comprises the action (action 110) of removing excess fluid from suction lipectomy fatty tissue.Excessive fluid can comprise blood and often comprise swelling fluid, and it can disturb the downstream processing action of relevant cell and be separated.In one embodiment, the sample of gravity settling or centrifugal segregation excess fluid is used can be divided into fat tissue layer and excess fluid layer, because fatty tissue has the density lower than excess fluid.Then fat tissue layer and excess fluid can be separated into different containers, so that from excess fluid isolated adipose tissue.Washing soln can be applied to fatty tissue to wash described tissue, described washing soln comprises such as salts solution, lactated Ringer solution, hanks' balanced salt solution or phosphate buffered physiological saline solution, and can repeat delaminating process to wash fatty tissue more up hill and dale and to remove excess fluid.In another embodiment, the strainer comprising net can be used to discharge excess fluid.Washing soln can be added into fatty tissue and use strainer to discharge to wash described tissue.This washing process can be repeated.In some embodiments, strainer can comprise such as, hole for about 30 microns (μm) to about 1 millimeter (mm), about 30 μm, about 50 μm, about 70 μm, about 85 μm, about 100 μm, about 120 μm, about 140 μm, about 200 μm, about 300 μm, about 500 μm, about 700 μm or about 1mm, aperture.In other embodiments, strainer can comprise the hole that aperture is about 70 μm to about 500 μm, such as about 70 μm, about 100 μm, about 140 μm, about 200 μm, about 300 μm or 500 μm.Or rather, strainer can comprise the screen filter that aperture is about 70 μm to about 200 μm, such as about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 140 μm, about 170 μm or about 200 μm.In another embodiment of the present disclosure, the aperture of strainer is less than described fatty tissue and is remained by strainer to make tissue.For effectively removing excess fluid, can apply washing action about 1 to about 10 times, such as once, twice, three times, four times, five times, six times, eight times or ten times, described washing action comprises to be added washing soln and removes washing soln.The volume of fat sample and add ratio between the volume being used for the washing soln at every turn washed can about 1: 0.2 and about between 1: 10, such as about 1: 0.2, about 1: 0.3, about 1: 0.5, about 1: 0.7, about 1: 1, about 1: 2, about 1: 3, about 1: 5 or about 1: 10.Such as, the 100ml suction lipectomy fatty tissue using tumescent liposuction technique art to collect can mix with the lactated Ringer solution of 100ml, and uses nylon wire to discharge, and described nylon wire has the hole that aperture is about 140 μm.This process can be carried out three times to complete the removal of excess fluid.In another embodiment, each washing action comprises and washing soln is added into sample and removes from sample, the volume of described washing soln is between 0.6 times and 4 times of sample volume, 0.6,0.8,1,1.2,1.5,1.8,2,2.5,3 or 4 times of such as sample volume, and wash action carry out once, twice, three times or four times.In another embodiment, excess fluid removal action may be combined with the action using gravity stratification, then uses strainer to discharge.In another embodiment, washing soln can be added into and mix with untreated fatty tissue to dilute excess fluid, then layering or use net formula strainer displacement fluids.Add washing soln can make layering or discharge more effective.

In another embodiment of the present disclosure, excess fluid removal action is by putting into the container with outlet and then discharging excess fluid to carry out when not using strainer by sample.In one embodiment, container can comprise the component controlled for fluid further, such as, press from both sides valve.For carrying out the removal of excess fluid, excess fluid can be discharged by outlet, and when sample approaches outlet, flow control member can be used export blockade.The size of outlet can be less than adipose tissue sample or outlet has the large-size allowing tissue sample to pass through.Described action can manually or by means of sensor be carried out, described sensor such as optical pickocff or infrared sensor, and it detects sample about outlet.Washing soln can be added into adipose tissue sample so that washing or cleaning sample, and excess fluid removal action can be repeated with clean adipose tissue sample.Second action (action 120) of the described method shown in Figure 1A is the fatty tissue that dissociates.Fatty tissue can use ultrasonic wave to dissociate.Fatty tissue can use the solution that dissociates to dissociate.The solution that dissociates can comprise the enzyme decomposing extra fat tissue cell's matrix.The solution that dissociates can comprise collagenase, proteolytic enzyme (protease), proteolytic enzyme (proteinase), neutral protease, elastoser, Unidasa, lipase, trypsinase, release enzyme, DNA enzymatic, deoxyribonuclease, stomach en-or its mixture.The solution that dissociates can comprise concentration between 0.1mg/ml and 10mg/ml, such as the collagenase of about 0.1mg/ml, about 0.2mg/ml, about 0.3mg/ml, about 0.5mg/ml, about 0.75mg/ml, about 1mg/ml, about 1.2mg/ml, about 1.5mg/ml, about 2mg/ml, about 3mg/ml, about 4mg/ml, about 5mg/ml, about 7mg/ml or about 10mg/ml.The solution that dissociates can comprise trypsinase.The solution that dissociates can comprise collagenase and deoxyribonuclease.The solution that dissociates can comprise collagenase, Unidasa and deoxyribonuclease.The solution that dissociates can comprise the Unidasa between collagenase between 0.2mg/ml and 5mg/ml, 0.1mg/ml and 4mg/ml and the deoxyribonuclease between 1 unit and 400 units, wherein per unit be defined as using DNA as substrate, under the Mg++ concentration of 4.2mM at pH5.0, the every ml of per minute produces the enzymic activity of the A260 change of 0.001 at 25 DEG C.The solution that dissociates can comprise calcium ion and/or magnesium ion further.The solution that dissociates can comprise concentration between 0.1mM and 10mM, the magnesium of such as about 0.1mM, about 0.2mM, about 0.3mM, about 0.5mM, about 0.7mM, about 1mM, about 1.5mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM or about 10mM or calcium ion.

After solution is dissociated in interpolation, fatty tissue can hatch certain for some time at a certain temperature (such as about 37 DEG C), such as about 5 minutes to about 30 hours.In the process of hatching, fatty tissue conciliates exsolution liquid intermittently and/or can continue mixing to be conducive to effective reaction.Fatty tissue dissociates action or hatch action and can carry out at 37 DEG C, continue about 10 minutes to about 120 minutes, such as about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes or about 120 minutes, with stirring gently continuously or intermittently the adipose tissue sample dissociated in solution, wherein frequently stirred than every 3 minutes, such as per second, every 2 seconds, every 3 seconds, every 5 seconds, every 10 seconds, every 20 seconds, every 30 seconds, every 45 seconds, every 60 seconds, every 90 seconds, every 120 seconds or every 180 seconds.

When dissociating release, metal ion chelation agent can be added carry out isolated metal ion as ethylenediamine tetraacetic acid (EDTA) (EDTA) and stop the activity of the enzyme dissociated in solution, and temperature can be reduced between about 4 DEG C and 30 DEG C, such as room temperature, about 25 DEG C, about 22 DEG C, about 18 DEG C, about 15 DEG C, about 12 DEG C, about 8 DEG C or about 4 DEG C.After hatching, temperature can remain on about room temperature, such as about 25 DEG C, or between 18 DEG C and 28 DEG C.In another embodiment, after action of dissociating, blood plasma, platelet rich plasma or serum can be added suppress the enzyme dissociated in solution.

Free grease, matrix fiber, fragment of tissue are removed in 3rd action (action 130) of the method for Figure 1A, and undesired cell, such as adipocyte.Adipocyte, matrix fiber and fragment of tissue can use net formula strainer substantially to remove, described net formula strainer comprises aperture between about 10 μm and about 70 μm, the hole of such as about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 50 μm, about 60 μm or about 70 μm.In another embodiment, the adipose tissue sample dissociated by aperture between about 20 μm and about 50 μm, the net of such as about 20 μm, about 22 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm and about 45 μm and about 50 μm.The filtrate by net formula strainer can be collected.Or, can use and centrifugally substantially remove adipocyte, matrix fiber and fragment of tissue.

4th, the 5th and the 6th action (action 140,150 and 160) of the method for Figure 1A comprises minimizing red blood corpuscle (RBC), concentrated relevant cell and washed cell respectively.The order of these three actions is interchangeable.Such as, in one embodiment, first cell can concentrate, wash, and then reduces RBC.In another embodiment, first cell can wash, and concentrates afterwards to it.In another embodiment, can wash and concentrating cells simultaneously.Concentrated action can advantageously be carried out in the application needing smaller size smaller.Such as, often carry out under study for action cultivating the cell be separated from fatty tissue.For obtaining higher inoculating cell density in cell culture flasks, and for increasing culture efficiency, can concentrating cells.The cell be separated also can be used for being transplanted to or being expelled in human body or animal, wherein often needs high cell concn to produce good result.Cell concentration also can have following advantage: substantially remove the reagent used in prior actions, the enzyme such as dissociated in solution, the growth capable of inhibiting cell or cause detrimental action when being transplanted in animal or human patient of described reagent.In one embodiment, microfluidic device can be used to carry out concentrated relevant cell and/or remove red blood corpuscle.Microfluidic device can be configured to realize the 4th, the 5th and/or the 6th action simultaneously.In addition, microfluidic device can be configured to remove lymphocyte to reduce immunoreactive potentiality.In another embodiment, concentrated action can use centrifugal realization.In another embodiment, concentrated action can use membrane filter to realize.In another embodiment, concentrated action can use tubular fibre, such as tubular fibre membrane filter to realize.In another embodiment, red blood corpuscle minimizing action can use globulolysis solution, such as ammonium chloride to realize, and in globulolysis solution, red blood corpuscle optionally dissolves.

6th action (action 160) of the embodiment shown in Figure 1A is washing relevant cell and/or is transferred in the damping fluid of needs by relevant cell.Centrifugal, buffer-exchanged and/or dialysis method can be adopted.Or microfluidic device also can be configured to carry out This move.This move further reduces the residual agent used in prior actions, and may be needs when relevant cell is ready to use in clinical transplantation.But, in some embodiments, action 140-160 can be omitted.

In an embodiment of the present disclosure, a kind of method comprises preconditioning fatty tissue, dissociate fatty tissue and the refining cell discharged.Figure 1B illustrates the schema of this embodiment of this method being generally designated as 200.In one embodiment, fatty tissue preconditioning action (action 210) comprises discharges waste streams, removal waste streams, removal excess fluid, cleaning adipose tissue sample, washing adipose tissue sample and/or chopping adipose tissue sample.When adipose tissue sample comprise be difficult to enzymic digestion or dissociate bulk time, chopping can be favourable.Fatty tissue preconditioning action can use disclosed in the disclosure and realize for the aspect and embodiment being separated non-fat cell mass from fatty tissue.Such as, fatty tissue preconditioning action can comprise and being retained in the first container by adipose tissue sample, and makes excess fluid if blood, tumescent solution or other body fluid are by arriving second container simultaneously.The reservation of adipose tissue sample can use the net in the first container to realize.In one embodiment, the aperture of net between about 70 μm and about 300 μm, such as about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 140 μm, about 170 μm, about 200 μm or about 300 μm.Or the reservation of adipose tissue sample can use and detect adipose tissue sample and realize about the detector of the position of the first container or sensor, and does not use the net in the first container.Fatty tissue preconditioning action can comprise further once or repeatedly adds and remove cleaning solution, to wash adipose tissue sample.Organize preconditioning also can use centrifugal realization, wherein adipose tissue sample is under centrifugal force separated with excess fluid and/or waste streams.Or, if fatty tissue is in for dissociate and under refining acceptable terms, fatty tissue preconditioning action can be omitted.

In one embodiment, fatty tissue dissociates under action (action 220) is included in the temperature being suitable for enzymic digestion, such as between about 32 DEG C and about 38 DEG C, hatch adipose tissue sample dissociating in solution, time length is between about 3 minutes and 20 hours, the described solution that dissociates comprises at least one enzyme, such as collagenase, proteolytic enzyme (protease), proteolytic enzyme (proteinase), neutral protease, elastoser, Unidasa, lipase, trypsinase, papoid, release enzyme, DNA enzymatic, deoxyribonuclease, stomach en-or its combination.In another embodiment, fatty tissue action of dissociating comprises and makes ultrasonic wave pass through adipose tissue sample.Dissociate in course of action at fatty tissue, cell discharges from adipose tissue sample.

Refining (action 230) of the cell discharged can comprise use strainer, net, tubular fibre, antibody, microfluidic device or centrifugal carry out cell concentration, cell washing, cellular segregation (cell separation), cellular segregation (cell isolation), fragment is removed, non-target cell is removed, red blood corpuscle reduces or the combination of action.For many application as point-of-care applications and rig-site utilization, may need within shorter for some time, within such as 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes or 120 minutes, carry out whole method of the present disclosure.

Many actions and embodiment for being separated non-fat cell mass from fatty tissue disclosed herein also can be used in research and pharmacology test macro.The separation of non-fat cell can be carried out, for physiology, metabolism and functional study or for carrying out drug test in pharmacology test macro.Many actions disclosed herein and embodiment also can be used in transplanted tissue's engineering.The cell using method disclosed herein and/or device to obtain can vitro culture to form the transplanting tissue of vitro culture.Separable adipose-derived stem cell is to produce functional cell and tissue.

An embodiment of the present disclosure be schematically show in Fig. 2 A and be generally designated as 300 sample processing device.Sample processing device 300 comprises sample surge chamber 310 and waste compartment 320.Sample surge chamber 310 comprises the first entrance 305 for receiving sample 340, described sample such as liposuction aspirates or the adipose tissue sample from lipsuction; For receiving the second entrance 315 of cleaning solution from cleaning solution source 350, described cleaning solution such as buffered soln, physiological saline solution etc.; For collecting the first outlet 325 regulating rear sample 360; And be connected to the second outlet 335 of waste container 320.Second outlet 335 can comprise component 345 and be connected to the fluid opened and closed between surge chamber 310 with waste container 320, and described component is valve, folder valve, stopcock etc. such as.Sample surge chamber 310 allows sample its excess fluid to be discharged in waste container, and uses cleaning solution to wash.Surge chamber can comprise sensor further, and it is preferably close to the second outlet 335, to detect the whether close outlet of the sample when excess fluid or cleaning solution are discharged in waste container.Surge chamber can comprise further and is placed on the first entrance 305 and second and exports strainer between 335, and described strainer is configured at sample washing, sample clean and removes in excess fluid process and retain fatty tissue.In some embodiments, strainer can comprise strainer, described strainer comprises aperture at about 30 μm and about between 1mm, the hole of such as about 30 μm, about 50 μm, about 70 μm, about 85 μm, about 100 μm, about 120 μm, about 140 μm, about 200 μm, about 300 μm, about 500 μm, about 700 μm or about 1mm.In other embodiments, strainer can comprise aperture and be about 70 μm to about 500 μm, the hole of such as about 70 μm, about 100 μm, about 140 μm, about 200 μm, about 300 μm or 500 μm.Strainer can comprise aperture and be about 70 μm to about 200 μm, the screen filter of such as about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 140 μm, about 170 μm or about 200 μm.In another embodiment, the aperture of strainer is less than fatty tissue sheet, makes tissue be remained by strainer like this.

Cleaning solution can enter surge chamber via flow rate control device 330, described flow rate control device such as peristaltic pump, and it controls the volume being added into the cleaning solution of surge chamber.Flow rate control device can comprise at least one valve and the changeable container of volume.Such as, flow rate control device can comprise stopcock 370 and syringe 380, as Fig. 2 B schematically shows.For distributing the volume measured, first stopcock switches to the position that entrance is connected with syringe fluid.Pull the piston of syringe with by fluid from entrance suction syringe.Then stopcock is switched to the position exporting and be connected with syringe fluid.Next, the piston of pushing syringe is to distribute syringe via outlet by fluid.Finally, switch cock valve is to be connected syringe with inlet fluid again, thus completes pumping circulation.Pumping circulation can be repeated until volume required cleaning solution is added into surge chamber.Flow rate control device also can comprise two vacuum breaker CV1, CV2 (namely allowing the valve that fluid flows in one direction) and syringe, as such as Fig. 2 C schematically shows.For distributing the volume measured, first pulling the piston of syringe and then promoting, to complete pumping circulation.First vacuum breaker (CV1) is configured to allow fluid to flow to syringe from entrance, and the second vacuum breaker (CV2) is configured to allow fluid to flow to outlet from syringe.Can pumping circulation be repeated, comprise and pull and promote piston with respectively by fluid suction with fluid is released syringe, until volume required cleaning solution is added into surge chamber.Or the bag that the syringe in the flow rate control device described in Fig. 2 B or Fig. 2 C can expand and shrink with or the container that volume can change in a controlled manner substitute.

Another embodiment of the present disclosure be schematically show in Fig. 2 D and be generally designated as 400 sample processing device.Sample processing device 400 comprises sample dissociation chamber 410 and cell refining plant 420.Dissociation chamber comprises the first entrance 405 receiving sample, the second entrance 415 receiving the solution that dissociates from source of solvent 430 of dissociating, and at least one connection with cell refining plant fluid exports 425.Dissociation chamber is configured to sample to be dissociated into little moiety, such as individual cells and minicell grumeleuse.The solution that dissociates can comprise the enzyme decomposing sample.Such as, the solution that dissociates can comprise collagenase, proteolytic enzyme (protease), proteolytic enzyme (proteinase), neutral protease, elastoser, Unidasa, lipase, trypsinase, release enzyme, DNA enzymatic, deoxyribonuclease, stomach en-or its mixture.Temperature can be controlled at such as about 37 DEG C, and sample in dissociation chamber and fluid can mix to be conducive to effective enzyme reaction and homogeneous dissociating.An embodiment of dissociation chamber is flexible pouch.Bag can rub, extrudes, rolls, shakes, shakes, portion extrusion and front and back release or otherwise stir and be conducive to mixing.The solution that dissociates also can comprise sanitising agent, as polysorbas20 or sodium lauryl sulphate.When use ultrasonic wave dissociates sample, the solution that dissociates can comprise the medium of effective conduct ultrasound.Dissociation chamber can comprise strainer, such as net or strainer.Strainer can be used for sample to remain on for efficient solution from position and/or be used for removing large fragment from the sample dissociated.In some embodiments, strainer can comprise strainer, described strainer comprises aperture for about 30 μm to about 1mm, the hole of such as about 30 μm, about 50 μm, about 70 μm, about 85 μm, about 100 μm, about 120 μm, about 140 μm, about 200 μm, about 300 μm, about 500 μm, about 700 μm or about 1mm.In other embodiments, strainer can comprise aperture and be about 70 μm to about 500 μm, the hole of such as about 70 μm, about 100 μm, about 140 μm, about 200 μm, about 300 μm or 500 μm.Strainer can comprise aperture and be about 70 μm to about 200 μm, the screen filter of such as about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 140 μm, about 170 μm or about 200 μm.In another embodiment, the aperture of strainer is less than fatty tissue sheet, makes tissue be remained by strainer like this.

Cell refining plant is connected to dissociation chamber.In some embodiments, sample processing device is included in the valve 435 between dissociation chamber and cell refining plant further.Valve can cut out and hatch sample for the solution that dissociates, and can open to allow discharged cell to enter cell refining plant.

Cell refining plant is configured to receive from dissociation chamber the cell and the refining cell discharged that discharge.In some embodiments, cell refining plant comprise be connected with dissociation chamber fluid via entrance 445 room, for gathering in the crops the outlet 455 of refining cell 440, and structure removes the strainer of large fragment in the sample dissociated.Strainer can comprise aperture between about 10 μm and about 100 μm, the strainer of such as about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 50 μm, about 70 μm or about 100 μm.Strainer also can comprise aperture between about 20 μm and about 60 μm, the net of such as about 20 μm, about 22 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 50 μm and about 60 μm.

Another embodiment of the present disclosure be schematically show in Fig. 2 E and be generally designated as 500 sample processing device.Sample processing device 500 comprises the first Room 510, waste container 520 and cell refining plant 530.As disclosed above, cell refining plant can comprise net.Preconditioning room and dissociation chamber can be served as in first Room, and in preconditioning room, sample can wash before dissociating, and in dissociation chamber, sample can be dissociated into less moiety, such as individual cells or minicell aggregate.First Room comprises the first entrance 505 for receiving sample and the second entrance 515 for receiving cleaning solution from cleaning solution source 350.In some embodiments, cleaning solution reconciliation exsolution liquid can enter the first Room via same entrance.In other embodiments, the first Room comprises the 3rd entrance 525 for receiving the solution that dissociates from source of solvent 430 of dissociating.First Room can comprise strainer further, such as net or strainer, described in the above paragraph about dissociation chamber and surge chamber.First Room is connected with waste compartment and cell refining plant fluid.Valve V1 and V2 such as pressing from both sides valve or clack valve can be comprised can be used for controlling fluid flow rate.Such as, in sample preconditioning process, valve V1 can open excess fluid and cleaning solution can be flowed into waste container from the first Room.V1 and V2 can close to allow to hatch sample with the solution that dissociates in sample dissociation process.After dissociating, V2 can open to allow the sample dissociated to be transferred to cell refining plant.

In embodiments more of the present disclosure, before being loaded into the first Room 510, by adipose tissue sample pre-heating to a certain temperature, such as 37 DEG C.Pre-heating can shorten treatment group tissue samples required time as to the process of adipose tissue sample.In other embodiment of the present disclosure, use the light of mean wavelength between 300nm and 700nm by adipose tissue sample photoactivation, be then loaded in the first Room 510.Photoactivation can increase the usefulness of cell in tissue sample.In another embodiment of the present disclosure, before being loaded into the first Room 510, use sonic treatment adipose tissue sample, namely sound wave makes to organize loose, thus causes tissue sample to be easy to dissociate.

Sample processing device can comprise further as previously discussed for controlling the flow rate control device 330 of the flow of the cleaning solution entering the first Room.Can also can be used for the flow controlling the solution that dissociates be injected in the first Room by the flow rate control device 330B similar to flow rate control device 330.In some embodiments, the solution that dissociates was loaded into syringe before injection first Room.

Sample processing device disclosed herein should be understood and can have change and combination.Such as, as shown in Figure 2 F, the fluid flow rate that the general embodiment being generally designated as the sample processing device of 500A can adopt two valves (V1, V2) to control between the first Room, waste container and cell refining plant.Flow rate control device can comprise two vacuum breaker (CV1, CV2) and volume measurement device 540, such as syringe.The solution that dissociates can be connected to the first Room via vacuum breaker CV3.

Schematically show another embodiment of the sample processing device of the present disclosure being generally designated as 500B in fig 2g, wherein use at least 3 stopcocks (SC1, SC2, SC3) to control fluid flow rate.Such as, in sample preconditioning process, excess fluid and cleaning solution can be transferred to waste container from the first Room.Stopcock (SC3) can cut off the flowing that (shut off) exits the first Room, to allow to hatch sample with the solution that dissociates in sample dissociation process.After dissociating, the first Room can be connected to cell refining plant by stopcock (SC3), to allow the sample dissociated to be transferred to cell refining plant.

Fig. 3 A illustrates the embodiment of the device schematically shown in Fig. 2 E.Described device comprises two plastic sheets being bonded together to be formed multiple room.Described device can by streamlined, be easy to use, safety and there is cost-benefit mode to be conducive to the method for fatty tissue process disclosed herein.Room 1 is the measuring chamber that can expand to a certain volume.Described room comprises entrance (port one) and outlet (port 2).Room 1 can be designed to include fluid in, and distributes the fluid of a certain pre-determined volume, thus controls to treat to be dispensed to the volume of room 2 for the fluid of fatty tissue process by entrance (port 3).Port 2 is connected by pipe string fluid with port 3, described pipeline can use folder valve, clack valve, stopcock, vacuum breaker or one or more other establish valve system clamp with make port 2 from port 3 disconnect fluid connect.For operation room 1, start most to turn off the connection between port 2 and port 3.Fluid is introduced room 1 via port one expand with the completely or partially room of making.Then, use such as spring pinchcock, folder valve, stopcock, vacuum breaker or one or more other establish valve system cut off port one.Next, the valve opened between port 2 and port 3 flows into room 2 to allow the fluid in room 1 when room 1 is shunk.The contraction of room 1 by use gravity, by syphonic effect or by other method as known in the art by external compression and/or compress described room and come.Room 1 can be positioned on higher than room 2 place, to be conducive to using gravity to dispense fluid to room 2.The fluid transfer of predetermined amount to room 2, is determined described amount by the difference of expanding between volume and retraction volume of room 1 by this process.If need the fluid of smaller size smaller, the volume controlling the fluid entering and/or exit room 1 partly can be compressed, extrudes and/or be clamped in room 1.Or a part of fluid distributed wherein only partly can be shunk in room 1.If need comparatively large vol, can repeat to expand-contraction process is for several times until by volume required fluid transfer to room 2.

Room 1, port one and port 2 can be the embodiment of the flow rate control device schematically shown in Fig. 2 B or 2C.

Port one and port 2 can comprise vacuum breaker, and described vacuum breaker is also called that only permission fluid enters and exit the check valve of room 1 respectively.The action of fluid of measuring and distribute setting volume becomes very simple: decompressing to room 1 allows fluid to enter via port one and fluid is released via port 2 by pressure space 1.

Or room 1 can be storing chamber, the solution of its pre-packaged needed for sampling process.Such as, lactated Ringer solution, balanced salt solution, physiological saline solution, the solution that dissociates, washing soln, cleaning solution, solution containing ethylenediamine tetraacetic acid (EDTA) (EDTA) and/or enzyme solution can be packaged in the part as described device in room 1.

In another embodiment, measuring chamber can comprise the syringe of piston, the fluid that described syringe carrys out suction by pulling and promote piston and/or distributes pre-defined volume.In another embodiment, measuring chamber can comprise the flexible pipe line be arranged on peristaltic pump, wherein uses peristaltic pump to control fluid flow rate.

Room 2 can be the embodiment of the first Room 510 schematically shown in Fig. 2 E.Room 2 can be fatty tissue washing chamber, and it comprises one or more entrance and exit.Dissociation chamber also can be served as in room 2, and can comprise at least one net further, such as, comprise the filter mechanism of screen cloth, semi-permeable membranes and/or porous or microporous membrane.Adipose tissue sample, the liposuction aspirates tissue of such as liposuction aspirates or fat can be introduced or be loaded in room 2 via entrance (port 4).Excess fluid from sample can be entered in waste collection room (room 3) by net via joint 1.Washing soln can be added into room 2 with washing and/or cleaning sample.Can washing soln be measured via room 1 and be dispensed in room 2.Solution in order to processing sample can be added to change sample.Hybrid component can be applied to make cleaning and to wash more effective to room 2.Such as, can rub, extrude gently, shake, shake, portion extrusion and front and back release or otherwise teeter column 2 be conducive to mixing.Then waste streams can be drained into waste collection room (room 3).Can use in mixing process and establish the outlet of valve member close chamber 2 (joint 1 and 2) to mix discharging to allow before waste streams between washing soln with sample thorough.Diagrammatic clip such as clip 1, clip 2 and/or clip 3 can clamp each room and the fluid of closing between each room connects in application drawing 3F.Washing process can repeat for several times, such as 2,3,4 or 5 times.In washing action, adipose tissue sample can be retained in room 2.For the adipose tissue sample comprising fritter, room 2 can comprise net or membrane filter carrys out keeping sample effectively.Multiple net can be incorporated to and/or filter layer (net 1 and net 2) provides the fatty tissue of hope to retain.

After washing, the solution that dissociates can be added into room 2, to dissociate adipose tissue sample release cells.The temperature that heating, cooling and/or temperature controlling system can be used the temperature of room to be set in a certain optimization is dissociated to be conducive to sample.Such as, described device can be placed in couveuse, water-bath and/or be placed to and contact with temperature-constant plate, temperature is maintained at about 37 DEG C being used for best enzymic digestion.The solution that dissociates can comprise one or more enzymes to divide to unhitch forms matrix, extracellular matrix etc.Such as, can at 37 DEG C, collagenase be used to carry out decomposes collagen fiber.Other reagent also can be used to come, for fatty tissue digestion, to comprise proteolytic enzyme (protease), proteolytic enzyme (proteinase), trypsinase, Proteinase K, lyase, enzyme, solvent soln, Unidasa, lipase, trypsinase, release enzyme, DNA enzymatic, deoxyribonuclease, stomach en-or its mixture.The outlet (joint 1 and/or joint 2) of dissociation chamber (room 2) can be closed in digestive process.Can rub, extrude gently, shake, shake, portion extrusion and front and back release or otherwise teeter column 2 be conducive to mixing and promote that effective fatty tissue dissociates.When digestion action completes, joint 2 can be opened and exit dissociation chamber to allow the cell discharged.At least one net in room 2 can be used for removing or retaining large stretch of fragment.One or more continuous print adding washing soln that comprises can be applied and wash action (chase wash act), to wash the rear potential cell be trapped in room 2 of digestion.

Sample processing device shown in Fig. 3 A can comprise fragment further and remove room (room 4), and it can comprise net, membrane filter and/or another debris removal mechanism (such as net 3).The sample dissociated can be transferred to fragment and remove room, wherein can remove bulk, unwanted cells as adipocyte and/or fragment from sample.Room 4 also can be used as sample storage chamber, wherein holds the cell that discharges until use further.The cell discharged can be collected via port 5.Room 4 can be the embodiment of the cell refining plant 530 schematically shown in Fig. 2 E.

Fig. 3 B and 3C illustrates that net is incorporated into the embodiment in room 4.Net collapsible, to be clipped between two flexible sheets and to weld, glued joint, seal or otherwise merge with forming chamber 4.Similarly, net or membrane filter can be incorporated in room 2.Fig. 3 D and 3E comprises decomposition view and the cross sectional view of a part for room 2 respectively, and it illustrates in room 2 can use one or more net.The net that room 2 and/or room 4 comprise can comprise and flows through the path of room 2 and/or 4 and the hole diminished gradually along fluid.Such as, the normal pore size of net 1 (Fig. 3 D) can be about 100 μm to about 900 μm, and the normal pore size of net 2 can be about 50 to about 200 μm, and the normal pore size of net 3 (Fig. 3 C) can be about 10 μm to about 60 μm.

The concrete structure that the disclosure is not limited to the embodiment shown in Fig. 3 A-3F should be understood.Embodiment of the present disclosure can comprise than the more or less room shown in Fig. 3 A-3F.Can adopt the room with difference in functionality, and these rooms can be configured to reach the specific tasks comprising a series of concrete actions.Such as, described room can have and includes but not limited to following function: sample collection, washing, cleaning, layering, mixing, heating, cooling, filtration, digestion, store, establish valve, cubing, pumping, fluid transfer and manipulation, cell marking, sample preparation, dissociate, waste streams is collected, grumeleuse is removed, fragment is removed, dissolve, concentrated, polymerase chain reaction (PCR), hatch, hybridize, cell cultures, cell amplification etc.

Two plastic sheets are used to form in Fig. 4 another embodiment of the sample processing device of the present disclosure being generally illustrated as 600.Room 1 is sample washing and dissociation chamber, and it comprises three ingress ports (port one, port 2 and port 3), net (net 1) and two outlet connections (joint 1 and joint 2).Room 2 is grumeleuse minimizing room, and it comprises inlet attack (joint 2), outlet/inlet attack (joint 3) and net (net 2).Room 3 optionally comprises net (net 3), its can be for separating of cell and the reservoir removed of further fragment, or refine room for cell.Room 4 is waste solution collecting chamber, and it comprises the source line (joint 1) washing with sample and be communicated with the outlet connection fluid of dissociation chamber (room 1).

Fig. 5 A and 5B illustrates another embodiment comprising the room 610 of two nets of the present disclosure.These two nets are configured to substantially not overlapping.Each net folding is also clipped between two flexible outer plates.This embodiment can have the advantage being comparatively easy to manufacture, because only have two layers of mesh must merge between sheet.

Fig. 6 A and 6B illustrates another embodiment of room 620 of the present disclosure, and it comprises at least one net do not folded be clipped between two flexible outer plates.Described net is positioned between ingress port and outlet port, and is constructed such that the fluid entered from ingress port must arrive outlet port by described net.Ingress port and outlet port are in the opposite side of net.This configuration can have the advantage being easy to manufacture, because only have one deck net must be sealed between two sheets.

Fig. 7 illustrates another embodiment of room 630 of the present disclosure, and it comprises the pleating net be clipped between two flexible outer plates.This structure can have the advantage that web area increases.

Fig. 8 A and 8B illustrates another embodiment of room 640 of the present disclosure, wherein net folding and along sealing edge sealing to form pouch before being incorporated into room.Such as, along fold line a slice plastic wire, polymeric amide net, and such as heated sealant or hight frequency welding can be used along two potted line sealings to form net pouch.Then net pouch can be positioned between two flexible plastic sheets, such as polyvinyl chloride (PVC) sheet, and uses such as heated sealant or radio frequency welding to seal along sealing edge, thus forming chamber.

Fig. 9 A and 9B illustrates another embodiment of room 650 of the present disclosure, wherein net folding, be clipped between two flexible plastic sheets, and to be sealed in room.Herein, room and net are constructed such that the sample entering room via ingress port can exit via outlet port 1, and not by net, and must net be passed through via a part of sample that outlet port 2 exits.It is vertical that the fold line netted is roughly.In another embodiment, fold line can be angled about vertical curve.

Institute's diagrammatic any one or multiple net structure can be utilized in Fig. 5 A-9B in the room of any sample processing device disclosed herein, such as, one or more in the room 2 or room 4 of sample processing device 500 and/or one or more in the room 1 of sample processing device 600, room 2 and/or room 3.

Figure 10 A and 10B illustrates the joint comprising at least one line sections between the two chambers, and described joint can be included in any sample processing device embodiment disclosed herein.By plastic sheet otch, and pipeline bridges to another room through described otch from a room.This structure can allow to establish valve member to access described pipeline.Such as, spring pinchcock or slide clamp can be adopted to open be connected with the fluid turned off by pipeline.Line segments can comprise the soft and stretch section of flexibility (pipeline 2 in Figure 10 A and 10B) further, to be conducive to reliable clamping.This can be favourable when using folder valve.Can be manual, pneumatic or with solenoid-actuated folder valve.When needed, flexible pipe line also can be conducive to wriggling pumping.

The embodiment of sample processing device disclosed herein can comprise other parts further, as diagrammatic in Figure 11 those (such as, the sharp shaped material of washing soln bag can be inserted, one or more spring pinchcock, Y shape insert position, sharp shaped material port and/or the female Luer for being connected to syringe), and/or can be used as and be connected to other module compared with the integral part of Iarge-scale system.Barrier film, injection port, sharp shaped material port, valve, vacuum breaker, pipe, adapter, female Luer, cloudy Luer lock, positive Luer lock, syringe, stopcock and/or other connection mechanism can be adopted to the part of the embodiment of the present disclosure that interconnects and other parts.

Another embodiment of the present disclosure be generally be illustrated as in Figure 12 700 sample preparation apparatus, it comprises sample dissociation chamber (room 1), waste container (room 2) and cell and refines room (room 3).Room 1 optionally comprises the first filter screen to be conducive to sample washing, cleaning and preconditioning.Net comprises the hole of aperture between about 20 μm and about 600 μm.For process suction lipectomy sample, the aperture of net can preferably between about 40 μm and about 200 μm, such as about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 140 μm, about 170 μm or about 200 μm.The fluid at stopcock control port 5 place connects, and described port dissociates and hatches in process can close at sample, can be connected to room 2 in sample washing process via port 7, and can be connected to room 3 for collecting the cell discharged via port 6.Room 3 can comprise the second filter screen, and its aperture is between about 15 μm and about 150 μm.Second net removes fragment and the refining cell discharged from the sample dissociated, and described cell can be collected at port 8 place.Device can comprise the opening receiving sample further, such as port 4 (it can comprise sharp shaped material port); Receive the entrance of cleaning solution, such as port one (it can comprise sharp shaped material); And at least one is for receiving the entrance of the solution that dissociates, such as port 2.

Another embodiment of the present disclosure be generally be illustrated as in fig. 13 800 a kind of sample preparation apparatus, it comprises and is bonded together to form two piece of flexible material, the such as plastics that sample dissociation chamber (room 1), waste container (room 2) and cell refine room (room 3) with pre-qualified pattern.Room 1 can comprise the first net (net 1) to be conducive to sample washing, cleaning and preconditioning.Room 3 can comprise the second net (net 2) that aperture is less than the aperture of the first net.Fluid between stopcock 1 watch-keeping cubicle 1, room 2 and room 3 connects.Room 1 comprises ingress port (port one), and it comprises the joint that can be conducive to receiving sample from syringe, and described syringe such as has the syringe of catheter tip.Room 1 comprises another port (port 2) further, and it is connected to the stopcock manifold comprising stopcock 2 and stopcock 3.Cleaning solution, such as lactated ringer's inj, can be connected to sample preparation apparatus via sharp shaped material.Syringe 1 can serve as flow rate control device together with stopcock 2, and described flow rate control device allows the cleaning solution of limited amount to be added into room 1.The solution that dissociates can be loaded in syringe 2, and is added into sample preparation apparatus via stopcock 3.The solution that dissociates can be loaded in syringe 2 in a concentrated form, and uses cleaning solution to reconstruct normal working concentration.Room 3 comprises outlet port, wherein can collect discharged cell.In some embodiments, the pressure increasing outlet port place is needed.Room 3 can be enclosed in pressurized compartment (room 4) in a gas-tight manner.Room 4 comprises pressure port, and wherein can apply the fluid pressurizeed, such as pressurized air, directly pressurizes to the fluid in room 3 with the flexible sheets by room 3.The embodiment of Figure 13 B diagram room 3, and Figure 13 C illustrates the embodiment of the pressurized compartment comprising room 4.By two flexible sheets being bonded together forming chamber 3 (Figure 13 B) in pre-qualified position.Otch (otch 1) is done, to allow another sheet enclosed chamber 3 and forming chamber 4 in described.

Another embodiment of the present disclosure comprises downstream processing unit (in Figure 14 institute diagrammatic DPU 1000), its can process further, refine, cultivate, increase and/or analyze handled by fatty tissue and/or the cell that is separated.Downstream processing unit can be configured to be conducive to such as one or more following functions: sample washs, sample concentration, sample separation (sample separation), example enrichment, sample separation (sample isolation), buffer-exchanged, sample marks, sample changes, filter, magnetic mark, magnetic resolution, polymerase chain reaction (PCR), antibody interacts, antibody is used to carry out affinity capture, cell imaging, enzyme-linked immunosorbent assay (ELISA), prepared by albumen, protein purification, protein enrichment, mass spectrum, high performance liquid chromatography, flow cytometry, cell sorting, functional examination, cell cultures, cell amplification, cytodifferentiation, immunological classification, transverse flow measures, fluorescence in situ hybridization, thymus nucleic acid (DNA) is hybridized, Yeast Nucleic Acid (RNA) is hybridized, thymus nucleic acid (DNA) reacts, Yeast Nucleic Acid (RNA) reaction etc.

Downstream processing unit can comprise microfluidic elements, and described microfluidic elements comprises at least one microfluidic device.Microfluidic device can comprise at least one and be less than about 1mm, such as the channel size of about 0.95mm, about 800 μm, about 600 μm, about 500 μm, about 400 μm, about 300 μm, about 200 μm, about 150 μm, about 100 μm, about 80 μm, about 60 μm, about 50 μm, about 40 μm, about 30 μm, about 20 μm and/or about 15 μm.Microfluidic device also can comprise the passage of the degree of depth with at least one substantially constant.Such as, microfluidic device can comprise about 1mm, about 800 μm, about 600 μm, about 500 μm, about 400 μm, about 300 μm, about 200 μm, about 150 μm, about 100 μm, about 80 μm, about 60 μm, about 50 μm, about 40 μm, about 30 μm, about 20 μm or about 15 μm of dark passages.The channel depth of microfluidic device can in 20% of the nominal path degree of depth.Microfluidic device can comprise passage on a surface substantially further, and described surface can be substantially smooth or bending.Microfluidic device also can comprise the passage be formed on one or more flat surfaces.

Microfluidic device can use micro production, nanometer to make and/or micro-processing technology is formed, and described technology includes but not limited to photoetching, etching, reactive ion etching, dark reactive ion etching, Wet-type etching, the marking, injection moulding, embossing, soft embossing, stereolithography, shaping, soft lithographic, anode linkage, supersonic bonding, self assembly and/or other manufacturing technology known in the art.

The embodiment of microfluidic elements of the present disclosure can comprise device disclosed in the following: International Application Serial No. PCT/US10/061866, international publication WO 2011/079217 A1, U.S. Patent number US 7, 150, 812 B2, U.S. Patent number US 7, 735, 652, U.S. Patent number US 8, 021, 614, U.S. Patent number US 8, 186, 913 B2, U.S. Patent Application Publication No. US 2012/0063664 A1, U.S. Patent Application Publication No. US 2011/0294187 A1, it is all incorporated herein by reference for all objects, described device adopts Dean stream, inertia force, centrifugal force, determinacy transversal displacement, post array, bar array, clamping stream, magnetic texure, antibody component, cell capture part, albumen catches part, thymus nucleic acid (DNA) part, Yeast Nucleic Acid (RNA) part, filter, tangential flow filtration, focusing ultrasonic wave, clamping stream etc.

It should be noted that, embodiments more of the present disclosure, particularly disclosed those of microfluidic device of being incorporated in international publication WO2011/079217 A1, there is provided the device of anti-Severe blockage and contamination, Severe blockage and contamination are stop the serious problems using microfluidic device to carry out the fatty tissue for tangential flow filtration digestion up to now always.

Another embodiment of the present disclosure comprises hollow fiber unit, the cell be separated with concentrated and/or washing.

In another embodiment of downstream processing unit comprising microfluidic device, microfluidic device washed cell and remove undesired reagent.Downstream processing unit can comprise buffer solution inlet, carrys out washed cell to introduce buffered soln.The microfluidic device that cell washing also can use design to carry out analyzing realizes.

In another embodiment of the present disclosure, downstream processing unit comprises microfluidic device, enzyme concn reduction in the output of downstream processing unit is greater than about 10 times by described microfluidic device, such as enzyme concn reduction about 10, about 20, about 30, about 40, about 50, about 70, about 100, about 150, about 200, about 400, about 500, about 750, about 1,000, about 2,000, about 5,000, about 10,000, about 20,000, about 50,000, about 100,000, about 200,000, about 500,000 or about 1,000,000 times.An embodiment that can realize the microfluidic device that described enzyme is removed is disclosed in international publication WO 2011/079217 A1, and wherein microfluidic device comprises post, and adopts at least one damping fluid stream (such as cleaning solution stream) to carry out washed cell.

In another embodiment of the present disclosure, downstream processing unit comprises microfluidic device, the enzyme introduced in the sample dissociation process being greater than 89% is removed by described microfluidic device, such as, eliminate about 90% of the enzyme introduced in sample dissociation process, about 95%, about 97%, about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.95%, about 99.98%, about 99.99%, about 99.995%, about 99.998%, about 99.999%, about 99.9995% or about 99.9999%.

In another embodiment of the present disclosure, downstream processing unit comprises microfluidic device, and the enzyme introduced in the sample dissociation process of about 100% is removed by described microfluidic device.

In another embodiment of the present disclosure, downstream processing unit comprises microfluidic device, described microfluidic device provides collagenase concentration to be less than about 0.1mg/ml, be such as about 0.09mg/ml, about 0.05mg/ml, about 0.03mg/ml, about 0.02mg/ml, about 0.01mg/ml, about 0.007mg/ml, about 0.005mg/ml, about 0.003mg/ml, about 0.002mg/ml, about 0.001mg/ml, about 0.0005mg/ml, about 0.0002mg/ml, about 0.0001mg/ml, about 0.00005mg/ml, about 0.00002mg/ml, about 0.00001mg/ml, the output of about 0.000001mg/ml or about 0.0000001mg/ml.

In another embodiment of the present disclosure, downstream processing unit comprises microfluidic device, and described microfluidic device provides the output being substantially free of the enzyme introduced in sample dissociation process.In another embodiment of the present disclosure, downstream processing unit comprises microfluidic device, and described microfluidic device provides the output not being contained in the enzyme introduced in sample dissociation process.

In another embodiment of the present disclosure, downstream processing unit comprises microfluidic device, and described microfluidic device provides the output being substantially free of the collagenase introduced in sample dissociation process.In another embodiment of the present disclosure, downstream processing unit comprises microfluidic device, and described microfluidic device provides the output not being contained in the collagenase introduced in sample dissociation process.

In Figure 15 A, 15B, 15C, 15D and 15E schematically figure solve generally be designated as respectively 910,920,930,940 and 950, the example of microfluidic device that can use in any one or multiple sample processing device disclosed herein.

Figure 15 A illustrates microfluidic channel 910, and it has the outlet of cell entry, buffer inlet, cell outlet and damping fluid.The width of microfluidic channel and/or the degree of depth so little (such as about 100 μm), form flow side by side and two the Laminar Flow streams mixed without remarkable convection current each other to make sample and damping fluid.Velocity of flow is configured to give time enough to unwanted particle (such as enzyme) and diffuses to damping fluid flow from cell flow.Because cell is much larger than unwanted particle, therefore its diffusion is that so consequently they are retained in cell flow slowly.Stream of cells and damping fluid flow through and exit microfluidic channel by the different mouths that exits, thus substantially remove unwanted particle.

Figure 15 B illustrates another microfluidic channel 920, and it has cell entry, two buffer inlet, cell outlet and two damping fluid outlets.Passage and flow velocity are configured to allow unwanted particle to diffuse to damping fluid stream from stream of cells, thus substantially remove unwanted particle.This configuration can have high removal efficiency, because by two damping fluid diffluences except unwanted particle.

Bar or post can be positioned in microfluidic channel, such as, as institute's diagram in institute's diagrammatic microfluidic channel 930 in Figure 15 C, to make cell and buffering flow stabilizes, promotion cellular invasion and/or to support microfluidic channel.

For the microfluidic device of dialysing can series configuration to form a cascade.An example shown in Figure 15 D, wherein removes the buffered soln carrying unwanted particle from damping fluid outlet 1, and can introduce fresh buffered soln via buffer inlet 2, substantially to remove remaining unwanted particle.

Figure 15 E illustrates another embodiment of microfluidic device 950, and it comprises passage and bar array in the channel.The flow velocity of the fluid in introduction passage and channel size are designed so that fluid stream is laminar flow.Usually, when the Reynolds number of fluid is less than about 1 in microfluidic devices, there is Laminar Flow and laminar flow stream.Bar array increases the effective diffusion coefficient of particle in damping fluid stream, and improves the removal efficiency of undesired particle (such as enzyme) from stream of cells.

In some embodiments, the damping fluid for the formation of the damping fluid stream in microfluidic device is cleaning solution.

In another embodiment, downstream processing unit comprises dialysis membrane.

Another embodiment of the downstream processing unit 1000 schematically shown in Figure 16 comprises microfluidic device unit, and described microfluidic device unit comprises at least one microfluidic device, its concentrated cell be separated; Collect reservoir, such as collecting bag with at least one, it is configured to the output of cell as microfluidic device of reception separation.Downstream processing unit can comprise the syringe being connected to described collection reservoir further.Use after microfluidic device processes, by output from collection reservoir suction syringe at sample.Downstream processing unit can comprise refuse reservoir further, such as litter bag.

In another embodiment of the present disclosure, downstream processing unit comprises multiple microfluidic device to realize the capacity required for output sample, turnout and the function that process large volume.

The transfer of fluid can use gravity, external pressure, vacuum, malleation, negative pressure, head height, pump (such as peristaltic pump), the mechanism constructing squeeze bag, the roller of squeeze bag, the plate of squeeze bag and/or other liquid transferring machine structure as known in the art to realize.In one embodiment, fluid can use syringe to shift.In another embodiment, fluid can use the external air pressure being applied to room to shift, and the room 4 of the bag (room 3) containing cell is such as closed in described room, as shown in figure 13.

In another embodiment of the present disclosure, downstream processing unit comprises the cell culture chamber for culturing cell.Cell culture chamber can use joint to connect, and described joint allows dismounting cell culture chamber.Cell culture chamber can be placed in couveuse, wherein can optimize temperature and the condition of Growth of Cells, such as, at the temperature of about 37 DEG C and under about 5% gas concentration lwevel.Cell culture chamber can comprise air permeable material further, such as filter membrane or silicone rubber film, thus allows gaseous interchange in cell cultivation process.

One or more rooms of sample processing device as disclosed herein can comprise a net, Multilayer Network and or a series of net (Fig. 5 B).In some embodiments, aperture (intermediate value of the opening in such as hole or mean sizes) for the net of fatty tissue process can at about 1 μm and about between 10mm, such as about 1 μm, about 3 μm, about 6 μm, about 10 μm, about 15 μm, about 25 μm, about 40 μm, about 70 μm, about 100 μm, about 140 μm, about 300 μm, about 700 μm, about 1mm, about 2mm or about 3mm.For from suction lipectomy separate tissue non-fat cell, screen distance can at about 10 μm and about between 2mm.In some embodiments, the net in about 40 μm, about 70 μm, about 100 μm, about 140 μm, about 250 μm and/or about 700 μm apertures can be adopted, such as polymeric amide (nylon) net.

Another embodiment of sample processing device of the present disclosure comprises one or more room, and described one or more room comprises two nets, and the second net is at the fluid communication downstream of the first net, and wherein the hole of the second net is significantly less than the hole of the first net.

Another embodiment of sample processing device of the present disclosure comprises one or more room, and described one or more room comprises two membrane filters.Second membrane filter can at the fluid communication downstream of the first membrane filter.The hole of the second membrane filter significantly can be less than the hole of the first membrane filter.

Another embodiment of sample processing device of the present disclosure comprises one or more room, and described one or more room comprises track etching membrane filter.

The embodiment of sample processing device as disclosed herein or its subassembly can use and include but not limited to following material build: thermoplastics, acrylonitrile-butadiene-styrene (ABS) (ABS), acrylplastics (PMMA), zylonite, cellulose acetate, cyclic olefine copolymer (COC), cyclic olefine copolymer (COP), ethylene-vinyl acetate (EVA), ethylene-vinyl alcohol (EVOH), fluoroplastics (PTFE, and FEP, PFA, CTFE, ECTFE, ETFE), ionomer, liquid crystalline polymers (LCP), polyoxymethylene (POM or acetal), polyacrylic ester (acrylplastics), polyacrylonitrile (PAN or vinyl cyanide), polymeric amide (PA or nylon), polyamide-imide (PAI), PAEK (PAEK or ketone), polyhutadiene (PBD), polybutene (PB), polybutylene terephthalate (PBT), pla-pcl (PCL), polychlorotrifluoroethylene (PCTFE), polyethylene terephthalate (PET), polycyclohexylene's dimethylene ester (PCT), polycarbonate (PC), PHA (PHA), polyketone, polyester, polyethylene (PE), polyether-ether-ketone (PEEK), PEKK (PEKK), polyetherimide (PEI), polyethersulfone (PES), chlorinatedpolyethylene (CPE), polyimide (PI), poly(lactic acid) (PLA), polymethylpentene (PMP), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide (PPA), polypropylene (PP), polystyrene (PS), polysulfones (PSU), polytrimethylene's ester (PTT), urethane (PU), polyvinyl acetate (PVA) (PVA), polyvinyl chloride (PVC), polyvinylidene dichloride (PVDC), styrene-acrylonitrile (SAN) and/or acrylonitrile-butadiene-styrene (ABS) (ABS).For medical use, flexible plastic sheet such as polyvinyl chloride (PVC), urethane (PU), ethylene-vinyl acetate (EVA), polymeric amide (PA or nylon) can be used as sheet material.In some embodiments, product treatment unit as disclosed herein can comprise be bonded together and between define two flexible sheets of one or more room.In other embodiments, sample processing device as disclosed herein can comprise flexible sheets, and described flexible sheets is bonded to rigidity or semi-rigid material (in such as thick plastic sheet and/or above disclosed material any one or more) and defines one or more room between flexible sheets and rigidity or semi-rigid material.

In some embodiments, the thickness of sheet material can at about 0.1mm and about between 0.8mm, such as about 0.1mm, about 0.15mm, about 0.2mm, about 0.25mm, about 0.3mm, about 0.35mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm or about 0.8mm.In other embodiments, the thickness of sheet material can at about 0.2mm and about between 0.4mm, such as about 0.2mm, about 0.25mm, about 0.3mm, about 0.35mm or about 0.4mm.

The material of membrane filter and/or net can include but not limited to cellulose acetate (CA), glass microfiber (GMF), polyethersulfone (PES), polypropylene (PP), regenerated cellulose (RC), polymeric amide (PA or nylon), tetrafluoroethylene (PTFE) and/or poly(vinylidene fluoride) (PVDF).

In some embodiments, the thickness of net materials can at about 10 μm and about 1, between 000 μm, such as about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 150 μm, about 175 μm, about 200 μm, about 250 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm or about 1mm.In other embodiments, the thickness of net materials can between about 50 μm and about 300 μm, such as about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 125 μm, about 140 μm, about 160 μm, about 180 μm, about 200 μm, about 220 μm, about 250 μm, about 275 μm or about 300 μm.

Standard plastic manufacturing technology can be used to build embodiment of the present disclosure, and described technology includes but not limited to that adhesives that Plastic Welding, heat-sealing, injection moulding, embossing, glue bonding, UV-light (UV) solidify, solvent bonding, hot gas welding, free-hand welding, speed most advanced and sophisticated to be welded, extruded welding, contact bonding, hot plate welding, hight frequency welding, radio frequency welding, injection moulding welding, ultrasonic welding, friction welding, spin welding, laser welding and/or solvent welding.

Embodiment of the present disclosure, such as, in Fig. 2 A-2G arbitrary sample processing device schematically illustrated, can use the ratio-frequency welding of thermal plasticity slice to fetch structure.Welding mould can be made of metal, such as aluminium, brass or stainless steel.Folding mesh sheet and pipeline part are placed between two thermal plasticity slices on welding mould.Then another welding die clamp residence is used to state thermal plasticity slice.When applying pressure, temperature and radio frequency power to welding mould, forming chamber.For sealing polyvinyl chloride (PVC) sheet with polymeric amide net, can be applied between about 25 DEG C and about 120 DEG C, such as about 25 DEG C, about 50 DEG C, about 60 DEG C, about 70 DEG C, about 80 DEG C, about 90 DEG C, about 100 DEG C, the temperature of about 110 DEG C or about 120 DEG C, at about 10psi and about between 600psi, such as about 10psi, about 20psi, about 30psi, about 40psi, about 50psi, about 60psi, about 80psi, about 100psi, about 150psi, about 200psi, about 300psi, about 400psi, the pressure of about 500psi or about 600psi, and about between 300W and 10kW, such as about 300W, about 500W, about 600W, about 700W, about 800W, about 900W, about 1kW, about 1.2kW, about 1.5kW, about 2kW, about 2.5kW, about 3kW, about 4kW, about 5kW, about 6kW, about 7kW, about 8kW, the radio frequency power of about 9kW or about 10kW.The time length that radio frequency power can apply between about 0.5 second and about 1 minute, such as about 0.5 second, about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 7 seconds, about 8 seconds, about 10 seconds, about 12 seconds, about 15 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds and about 60 seconds.Radio frequency power can be applied for several times, to form reliable sealing by identical or different intensity.For medical use, can manufacture described device in controlled clean environment, and use standard sterilising techniques to carry out sterilizing, described technology includes but not limited to γ irradiation, oxyethane (EO) sterilizing and ultraviolet (UV) irradiation.

In embodiments more of the present disclosure, sample preparation apparatus is aseptic.In embodiments more of the present disclosure, sample preparation apparatus is that single uses.In addition; in embodiments more of the present disclosure; sample preparation apparatus is to provide the barrier of protectiveness in fact of the environment of isolation; wherein sample protected avoiding such as is contacted or fluid contact with outside atmosphere and/or operator's direct physical by unfiltered airflow, thus minimizes or avoid to pollute and infection risk.

Should be understood that embodiment of the present disclosure can serve as protectiveness barrier, the described protectiveness barrier any direct physical substantially reduced or eliminated between adipose tissue sample with surrounding environment contacts, fluid connects and/or airflow connects.Can contact with sample direct physical, any part of the embodiment of fluid connects and/or unfiltered airflow connects device disclosed herein can be aseptic and/or single uses.Should be understood that the embodiment of device disclosed herein can substantially be protected sample to avoid Pollution risk and protect operator to avoid infection risk.

Should be understood that the disclosure realizes the design of extremely wieldy fatty tissue treatment unit.Should also be understood that the disclosure can remarkable simplified manufacturing technique and reduce the manufacturing cost of described fatty tissue treatment unit.Should be further understood that, embodiment of the present disclosure makes adipose tissue sample can use substantially by the device that sample is isolated with Compass Labs or hospital environment, and essentially no pollution and infection risk process safely.

Embodiment

Embodiment 1. is from people's suction lipectomy separate tissue non-fat cell

Use and comprise the method for action depicted in figure 1 and the device handler liposuction aspirates as shown in Fig. 3 A and 11.It is long that described device is about the wide and about 40cm of 25cm.Flexible plastic sheet is made up of polyvinyl chloride (PVC), and net is made up of polymeric amide (nylon).Net 1,2 and 3 has the normal pore size of about 140 μm, about 70 μm and about 35 μm respectively.Use tumescent liposuction technique art to collect people's liposuction aspirates of about 40ml from the donor of license, and processed in 24 hours from collection.Transport sample and at being stored in 4 DEG C before treatment.

First, apply clip 1 and 2 and close joint 1 and 2 (Fig. 3 F).The liposuction aspirates of about 100ml is added into described device via sharp shaped material port (Figure 11).Open clip 2 to be expelled under gravity in room 3 to allow the excess fluid comprising blood and tumescent solution.After substantially removing excess fluid, close clip 2 and open spring pinchcock 1 and enter measuring chamber (room 1) to allow the lactated Ringer solution of about 50ml.Close spring pinchcock 1 and open spring pinchcock 2.Use two dull and stereotyped extrusion chamber 1 so that lactated Ringer solution is transferred to room 2.Repeat this fluid measurement and transfer process until the lactated Ringer solution of about 100ml is added into room 2.Room 2 is used and rubs mixed lactic Ringer's solution and suction lipectomy matter sample gently, thus washing sample.Open clip 2 to discharge to allow waste streams.This washing action herein carries out three times.

By close clip 1 and 2 and will dissociate solution from Y shape insert position (Figure 11) be added into come in room 1 tissue to dissociate action.The solution that dissociates comprises and is dissolved in the collagenase of 200mg in 20ml lactated Ringer solution and the DNA enzymatic I of 50mg.More lactated Ringer solution is introduced room 1 to dilute the solution that dissociates.Then, the solution that will dissociate is transferred to room 2.Again lactated Ringer solution is introduced room 1 with washing chamber 1, and the solution that dissociated by remnants is transferred to room 2.Dissociate in course of action at tissue, the lactated Ringer solution of about 100ml is added into room 2.Device be placed in the couveuse of 37 DEG C and frequently rub, with effective mixed structure sample and the solution that dissociates.Action of dissociating is organized to carry out about 45 minutes to 60 minutes.

After dissociating, open clip 1 and remove room (room 4 of Fig. 3 A) to allow the cell discharged to enter fragment.Open clip 2 subsequently to allow sample by net 3.In This move process, substantially remove fragment of tissue and adipocyte.

Then, the solution feed institute's diagram and the microfluidic device 1100 be disclosed in International Application Serial No. PCT/US10/061866 to Figure 17 that will come from room 4 under gravity, described international application is all incorporated herein by reference for all objects.Described microfluidic device comprises about 110 modules be arranged on the flat surfaces of substrate.Each module comprises the post that about 900 are configured to four rows.The degree of depth of microfluidic channel is about 30 μm.

The output of microfluidic device shown in Figure 18 A and 18B.Concentrated non-fat cell is collected in (Figure 18 A) in karyocyte product section substantially.Compared with input, the volume of karyocyte product section reduces about 8 times.Then use acridine orange (2mg/l) to dye to cell, and use fluorescent microscope imaging.Image illustrates that adipocyte is not present in the output of products thing of microfluidic device substantially, and non-fat karyocyte is collected in karyocyte part substantially.Image also illustrates that cell is in good form and product output is substantially free of the cell of destruction.

The further cell characterizing use the methods and apparatus disclosed and be separated, for the monocytes counts using ADAM MC automatic mammalian cell counter to carry out the work of total monocytes counts and attachment.The output of room 4 has the liposuction aspirates about 6.0 × 10 of every ml process ⁵individual total karyocyte.Use is supplemented with the substratum of α-MEM as the monocytes counts of the work for adhering to of 10% foetal calf serum (FBS).The output of room 4 is sampled and cultivate 3 days at 37 DEG C, in the medium, at cell culture chamber (such as Tissue Culture Dish or cell culture flasks).After 3 days, discard substratum, and use Da Erboke phosphate buffered physiological saline solution washed cell culturing room.Then, use trypsinase that the cell being attached to culturing room is discharged from chamber surface, continue 3 minutes.The monocytes counts of the work of attachment is the liposuction aspirates about 1.5 × 10 of every ml process ⁵individual.

Embodiment 2. 1 kinds is for using the next method from people's suction lipectomy separate tissue non-fat cell of sample preparation bagging apparatus

Use as Figure 13 the sample preparation bagging apparatus described carry out handler's liposuction aspirates.Described device comprises sample dissociation chamber (room 1), waste compartment (room 2) and cell and refines room (room 3).Device is about that 24cm is wide, about 36cm is long, and is made up of polyvinyl chloride (PVC) sheet that two pieces of each about 0.3mm are thick.Sample dissociation chamber and the cell room of refining comprise the first nylon wire (net 1) and the second nylon wire (net 2).The aperture of the first net is about 125 μm, and the aperture of the second net is about 25 μm.First net aperture or can between about 70 μm and about 160 μm, such as about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm or about 160 μm.Second net aperture or can between about 20 μm and about 50 μm, such as about 20 μm, about 22 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm or about 50 μm.

Use tumescent liposuction technique art from the donor collector liposuction aspirates of license, and processed in 6 hours from collection.Transport sample and at being stored in 4 DEG C before treatment.

First, stopcock 1 is set in the position of junction chamber 1 and room 2.The solution stowage that dissociates of 100mg collagenase, 100mg Unidasa and 20,000U deoxyribonuclease will be comprised in syringe 2.Sharp shaped material is used sample preparation bagging apparatus to be connected to the cleaning solution bag comprising lactated ringer's inj.

Use there is catheter tip syringe by the suction lipectomy matter sample of about 75ml from port one flood chamber 1.

Using action carrys out clean suction lipectomy matter sample.For starting cycles of washing, stopcock 1 being set in off-position, being connected so that room 1 is disconnected fluid from room 2 and 3.Use syringe 1 and stopcock 2 as the lactated ringer's inj flood chamber 1 of flow rate control device by about 100ml, described flow rate control device uses the action of following series to carry out work: (a) switch cock valve 2 is to be connected to syringe 1 by cleaning solution; B () is by cleaning solution suction syringe 1; C () switch cock valve 2 is to be connected to room 1 by syringe 1; (d) by cleaning solution from syringe 1 flood chamber 1.Described sequence can be repeated until volume required solution is added into dissociation chamber.

Then, rub room 1 to mix cleaning solution and sample, and switch cock valve 1 is to be expelled to excess fluid (i.e. waste solution) in room 2.After discharge, the first cycles of washing completes.Cycles of washing repeats twice.

Washing action can comprise one or more cycles of washing, such as one, two, three, four or five circulations.

After washing, the solution that will dissociate is added into room 1.Also the cleaning solution of about 100ml is added into room 1.Then at 37 DEG C, hatch sample preparation bagging apparatus, continue the incubation time of about 60 minutes.Room 1 is rubbed in process with biased sample and the solution that dissociates at this moment.In some embodiments, also can use other incubation time, such as about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 75 minutes, about 90 minutes or about 120 minutes.

After hatching, enter room 3 from the respective cells of sample release by stopcock 1, and large fragment of tissue is caught by net 1 and is stayed in room 1.Large adipocyte and fragment are removed from the sample dissociated by net 2 in room 3 further.Then, outlet port in room 3 collects non-fat cell, comprises pericyte, the stem cell of adipose-derived and progenitor cell.

For concentrating residual red blood cells and the fragment of cell and the removal discharged, then, the cell of the release that the outlet port in the room 3 of sample preparation bagging apparatus is collected runs through as the first microfluidic device disclosed in international publication WO 2011/079217 A1.Described microfluidic device comprises layout 73 modules on a surface of a substrate.Each module comprises about 1,300 posts being configured to four rows.Microfluidic device comprises the passage of the substantially the same degree of depth (between about 35 μm and about 50 μm).In another embodiment, microfluidic device comprises the degree of depth between about 30 μm and about 80 μm, the passage of such as about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 60 μm, about 70 μm or about 80 μm.By the cell concentration of the output at microfluidic device about 3 times.In another embodiment of the present invention, microfluidic device can be used for cell concentration being greater than about 2.5 times, such as about 3 times, about 4 times, about 5 times, about 6 times, about 8 times, about 10 times, about 12 times, about 15 times, about 20 times, about 25 times, about 30 times, about 40 times, about 50 times, about 60 times, about 80 times, about 100 times or about 125 times.

For removing enzyme, by the second microfluidic device process cell disclosed in international publication WO 2011/079217 A1.Second microfluidic device comprises layout 83 modules on a surface of a substrate.Each module comprises the post that about 900 are configured to four rows.Cleaning solution stream is introduced each module, and cell is transferred in cleaning solution stream, be separated with enzyme.

The residual volume of collagenase after the second microfluidic device is measured in application enzyme-linked immunosorbent assay (ELISA).Result illustrates that collagenase concentration reduces 1,000 times, and refining cell contains the collagenase being less than 0.001mg/ml.

Although therefore described the many aspects of at least one embodiment, those skilled in the art should be understood and can easily expect various change, amendment, combination and improvement.This type of changes, revise, combine and improvement is intended to for a part of this disclosure, and is intended to be in spirit and scope of the present disclosure.Therefore, aforementioned explanation and figure are only exemplary.

Claims

1., for being separated an equipment for non-fat cell from adipose tissue sample, described equipment comprises:

First material piece;

Be bonded to the second material piece of described first material piece; And

Be defined in the multiple rooms between described first material piece and described second material piece, described multiple room comprises:

Sample dissociation chamber, it includes an inlet and an outlet;

Waste collection room, it comprises the entrance be communicated with the described outlet fluid of described sample dissociation chamber; And

Cell refines room, and it comprises the entrance be communicated with described sample dissociation chamber fluid, and outlet.

2. equipment as claimed in claim 1, wherein said sample dissociation chamber comprises screen filter further, and described screen filter comprises the hole of aperture between 70 μm and 300 μm.

3. equipment as claimed in claim 1, it comprises screen filter further, and described screen filter is included in described cell and refines in room, comprises the hole of aperture between 20 μm and 50 μm.

4. equipment as claimed in claim 1, wherein said sample dissociation chamber comprises the first screen filter further, described screen filter comprises the hole with the first aperture, and the wherein said cell room of refining comprises the second screen filter further, described second screen filter comprises the hole with the second aperture, and wherein said second aperture is less than described first aperture.

5. equipment as claimed in claim 1, it comprises the component that the described sample dissociation chamber of control, described waste collection room and described cell refine the fluid connection between room further.

6. equipment as claimed in claim 5, the described component wherein controlling fluid connection comprises stopcock.

7. equipment as claimed in claim 1, it comprises flow rate control device further, described flow rate control device is configured at least one of conciliating in exsolution liquid by cleaning solution and is incorporated in described sample dissociation chamber, and has the outlet be communicated with described sample dissociation chamber fluid.

8. equipment as claimed in claim 1, it comprises further executes stressed component for of refining in room described sample dissociation chamber and described cell.

9. equipment as claimed in claim 1, it comprises downsteam processing facilities further, the described outlet fluid that described downsteam processing facilities and described cell refine room is communicated with and comprises at least one microfluidic device, described microfluidic device is configured to the fluid exported from the described cell room of refining to be divided into the first solution and the second solution, described first solution has one or more relevant cells of the first concentration, described second solution has one or more relevant cells described of the concentration being less than described first solution, wherein said relevant cell comprises the non-fat cell be separated from adipose tissue sample.

10. an aseptic and fatty tissue treatment system for substantial barrier, it comprises:

Organized processing room, it comprises entrance, outlet, and at least one screen filter, and described screen filter is arranged between the described entrance of described organized processing room and the described outlet of described organized processing room;

Waste collection room, itself and described organized processing room are included in same enclosed space, and described waste collection room comprises the entrance be communicated with the described outlet fluid of described organized processing room; And

Fragment removes one in room and sample collection room, and described fragment is removed room and comprised debris removal mechanism, and described sample collection room and described organized processing room are included in same enclosed space, and are communicated with described organized processing room fluid.

11. 1 kinds of methods processing adipose tissue sample in tissue processing system, described method comprises:

Pending adipose tissue sample is incorporated in the first Room by the ingress port of the first Room;

Process the described adipose tissue sample in described first Room; And

Be transferred to described second Room by the outlet of cell by described first Room, the entrance by the second Room from described first Room, described second Room and described first Room are included in same enclosed space.

12. methods as claimed in claim 11, wherein process described adipose tissue sample and comprise the described adipose tissue sample that dissociates.

13. methods as claimed in claim 11, wherein process described adipose tissue sample and comprise from the described adipose tissue sample removal excess fluid described first Room.

14. methods as claimed in claim 11, wherein process described adipose tissue sample and comprise the described adipose tissue sample used in described first Room of cleaning solution washing.

15. methods as claimed in claim 11, wherein process described adipose tissue sample and comprise the described adipose tissue sample used in described first Room of cleaning solution washing, and use the described adipose tissue sample that the solution that dissociates comprising at least one enzyme dissociates in described first Room.

16. methods as claimed in claim 15, the wherein said solution that dissociates comprises collagenase.

17. methods as claimed in claim 15, the wherein said solution that dissociates comprises collagenase, deoxyribonuclease and Unidasa.

18. methods as claimed in claim 15, wherein use the solution described adipose tissue sample that dissociates that dissociates to carry out at about 37 DEG C.

19. methods as claimed in claim 11, it comprises the screen filter removal fragment using and be included in described second Room further.

20. methods as claimed in claim 19, the aperture of wherein said screen filter is between 15 microns and 100 microns.

21. methods as claimed in claim 11, it comprises further and is retained in described first Room by described sample, and waste streams is displaced through be included in screen filter in described first Room and described first Room the first outlet, enter in described 3rd Room by the entrance of the 3rd Room, described 3rd Room and described first Room are included in same enclosed space.

22. methods as claimed in claim 11, it comprises use microfluidic device enrichment non-fat cell mass further.

23. methods as claimed in claim 22, wherein said non-fat cell comprises stem cell.

24. methods as claimed in claim 11, it comprises further from described tissue processing system harvested cell.

25. methods as claimed in claim 11, it is included in further in downsteam processing facilities and processes described cell, described downsteam processing facilities is communicated with the outlet fluid of described second Room and comprises at least one microfluidic device, described microfluidic device is configured to described cell to be divided into the first solution and the second solution, described first solution has the non-fat cell of the first concentration, and described second solution has the non-fat cell of the concentration being less than described first solution.

26. methods as claimed in claim 24, the cell of wherein said results is vascular stroma part cell.